GNF351 is an AHR ligand. A competition AHR ligand binding assay was conducted as described under Materials and Methods. Mouse liver cytosol expressing humanized AHR was used in combination with increasing concentrations of the test compounds, along with the PAL, at a concentration of 420 pM. Samples were exposed to UV light, analyzed by tricine SDS-polyacrylamide gel electrophoresis, and transferred to a membrane. The radioactive bands were then excised and quantitated using a gamma-counter. Data represent the percentage of specific binding relative to the absence of a competitor ligand.

GNF351 exhibits a lack of AHR agonist activity at increasing concentrations. A cell-based luciferase reporter assay using human HepG2 40/6 cells was conducted. A, cells were treated with DMSO, TCDD (5 nM), or increasing concentrations of GNF351 for 4 h. B, quantitative reverse transcription-PCR was performed for RNA samples isolated from HepG2 40/6 cells treated with DMSO, TCDD (5 nM), or increasing concentrations of GNF351 for 4 h, and the expression of CYP1A1 levels, normalized to L13a levels, was examined. Each treatment was conducted in triplicate wells. Data represent the mean ± S.E.M. with statistically significant results indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001), which are relevant to the data sets as labeled in A and compared with control for B. The direct comparisons are shown by the presence of the same letter (a or b).

GNF351 antagonizes the DRE-mediated response in AHR in human and murine cells. Cells were treated with increasing concentrations of GNF351 in combination with 5 nM TCDD in stable human hepatoma-derived reporter cells (HepG2 40/6) (A) and with 2 nM TCDD in stable murine hepatoma-derived reporter cells (H1L1.1.1c2) (B) for 4 h, after which lysis buffer was added, and a luciferase assay conducted on the lysate. Each data set is the result of triplicate well treatments. Data represent the mean ± S.E.M. with statistically significant results marked (***, P < 0.001), which are relevant to the data sets as labeled. The direct comparisons are shown by the presence of the same letter (a).

GNF351 antagonizes the effect of an endogenous AHR agonist. HepG2 40/6 cells were treated with DMSO, I3S (100 nM), and increasing concentrations of GNF351 with 100 nM I3S. Luciferase readings were taken after cells were lysed. Treatments were conducted in triplicate wells. Data represent the mean ± S.E.M. with statistically significant results (***, P < 0.001) compared as labeled. The direct comparisons are shown by the presence of the same letter (a or b).

GNF351 acts as a DRE antagonist for up to 12 h. A, a time-course treatment was conducted using HepG2 40/6 cells. Cells were treated with DMSO, TCDD (5 nM), or GNF351 (100 nM) and TCDD (5 nM). Treatments were conducted at 4-, 8-, 12-, 16-, 20-, and 24-h time increments. At the end of each time point, cells were harvested using lysis buffer as described under Materials and Methods, and luciferase readings were conducted. Time points for DMSO control were taken at 8 and 24 h. B, data generated in A were replotted to determine the approximate ED50. The average of the TCDD values at each time point was determined, and each GNF351+ TCDD value was divided by the TCDD average to determine percentage of antagonism.

Agonist and antagonist properties of various AHR ligands. A, left, HepG2 40/6 cells were treated with 10 μM concentration of various AHR ligands to determine whether each compound displayed agonist activity at a higher concentration. Right, HepG2 40/6 cells were treated with 40 nM AHR ligands plus 5 nM TCDD (except vehicle) to test for antagonistic abilities of each compound at a lower concentration. B, left, H1L1.1.1c2 cells were treated with 10 μM of each AHR ligand to determine whether any exhibited agonist activity. Right, H1L1.1.1c2 cells were treated with 100 nM of each ligand in combination with 2 nM TCDD to determine their antagonistic ability. Each treatment is the result of triplicate wells. Data represent the mean ± S.E.M. with statistically significant results indicated (*, P < 0.05; ***, P < 0.001). The direct comparisons are shown by the presence of the same letter (a or b).

GNF351 antagonizes acute-phase response pathways. A, an acute-phase repression assay was conducted in Huh7 cells. Cells were pretreated for 1 h with the following compounds: DMSO (vehicle) alone, DMSO, GNF351 (1 μM), TCDD (10 nM), SGA360 (10 μM), and αNF (10 μM). A combination of IL-1β and IL-6 was then added to all wells, except the vehicle alone (control), at a concentration of 2 ng/ml for each cytokine, and the treatment was continued for an additional 6 h. B, a mouse ear edema assay was conducted as described under Materials and Methods, and data were generated from three 6-week-old male C57BL6/J (wild type) mice. Mice were treated with vehicle (acetone), TPA, GNF351, SGA360, or combinations of these compounds for 6 h. Data represent the mean ± S.E.M. with statistically significant results compared as indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The direct comparisons are shown by the presence of the same letter (a or b).